J O U R N A L OF ULTRASTRUCTURE RESEARCH 60, 4 4 - 5 1
(1977)
A Freeze-Etch and Thin-Section Study of Mycoplasmas in Vinca rosea Phloem E. J. BRAUN Department of Plant Pathology, Cornell University, Ithaca, New York 14853 ~Received November 12, 1976, and in revised form, March 7, 1977 Mycoplasmas in the phloem of V. rosea were examined in thin sections and freeze-etchings. The plasmalemma of the parasite possessed evenly distributed particles on both fracture faces; particie diameters ranged from 8.7 to 13.2 nm. Vesicles in some mycoplasmas were found, in serial sections, to be wholly intracellular. Vesicles occurred most frequently in mycoplasmas thought to be senescent (characterized by swollen cells with very electron-lucent cytoplasm and a few nucleic acid strands). The fracture faces of the vesicular membranes lacked particles, and irregularly shaped regions 16 to 24 nm in diameter were demarcated by furrows. The functional significance of this unusual membrane structure was not determined.
Plant pathogenic mycoplasmas are transmitted by phloem-feeding insects and are confined to the phloem tissue in infected plants. Intracellular vesicles have been seen in many plant pathogenic mycoplasmas and they have been described in some detail by Hirumi and Maramorosch (1973). These authors, who worked with the aster yellows agent, referred to such structures as vacuoles. The present study was undertaken to gain more information regarding the structure of plant pathogenic mycoplasmas, particularly that of their intracellular vesicles.
ferred to a refrigerator (4°C) for an additional 5 hr. After washing in buffer, the material was postfixed in 1% OsO~ in the same buffer for 12-18 hr at 4°C. Tissue was dehydrated in an acetone series and embedded in Epon-Araldite. Thin and thick (0.5 #m) sections were made with glass knives on a LKB-Huxley ultramicrotome. Serial thin sections were picked up on 100 mesh Formvar-coated grids. Freeze-etching. Samples comparable to those used for thin sectioning were excised in a similar manner and fixed in 3% glutaraldehyde in 0.08 M phosphate buffer (pH 7.0) at room temperature for 1 hr. They were then washed in buffer, infiltrated with 20 or 30% glycerol, and frozen in liquid Freon-22 cooled in liquid nitrogen. The specimens were fractured at -101°C in a Balzers freeze-etching machine (BA 360M) and shadowed with platinum and carbon according to the method of Moor and Mulethaler (1963). Replicas were cleaned with 70% H2SO4 (18 hr), 40% chromic acid (18 hr), and 70% H~SO4 (6 hr). Before and after the final H2SO4 treatment, replicas were transferred to bleach for 1-2 hr. The replicas were picked up on Formvar-coated grids. The terminology of Branton et al. (1975) has been used in identifying fracture faces. The fracture face of the membrane which is closest to the cytoplasm is termed the protoplasmic fracture face (pf) and that closest to the extracellular or endoplasmic space is termed the extracellular or endoplasmic fracture face (ef). Particle size and density on membrane fracture faces of the mycoplasmas were determined by taking data from 10 protoplasmic and 10 extracellular fracture faces. Maximum shadow width of 12 randomly selected particles were measured on each fracture face. Density was determined by counting particles within three randomly selected 1 cm ~ areas on each
MATERIALS AND METHODS Mycoplasmas were transmitted via dodder (Cuscuta epithymum Murr.) from phloem necrosis-diseased American elm (Ulmus americana L.) seedlings to periwinkle plants (Vinca rosea L.). Samples (petioles and axillary shoots) were taken from V. rosea plants showing advanced stages of symptom development characterized by extensive proliferation of axillary shoots, stunted, chlorotic leaves, and cessation of flowering. Samples of healthy tissue (petioles and midribs) were obtained from V. rosea plants grown from seed. Both diseased and healthy plants were grown under greenhouse conditions. Thin sectioning. Samples were excised in a petri dish containing 3% glutaraldehyde in 0.08 M phosphate buffer (pH 7.0). The central portion of each sample was transferred to fresh fixative and evacuated in a desiccator for 5-10 min. After 1 hr at room temperature the material in fixative was trans44 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
MYCOPLASMAS IN fracture face. All measurements were made on micrographs at a magnificationof x 90 000. Replicas and thin sections were observed and photographed with a Philips EM 201 at 80 kV; 0.5 t~m sections were viewed at 100 kV. RESULTS In V . r o s e a the phloem occurs in bundles or strands consisting of sieve-tube elements, companion cells, and phloem parenchyma cells. Phloem develops both externally and internally with respect to the xylem. Structurally, the bundles of external and internal phloem are very similar and no attempt was made to differentiate between them in this study. Phloem was recongized in freeze-etch preparations by its spatial relationship to the xylem and because its cells are narrower t h a n those of the surrounding vascular parenchyma and cortex. Sieve-tube elements can be distinguished from the surrounding cells by several criteria. In healthy plants the lumen of the sieve element Was always free oforganelles (Figs. 1 and 2). Mitochondria, plastids, and endoplasmic reticulum were confined to the parietal region of the sieve element protoplast. Invaginations of the plasmalemma (Fig. 2) were common in sieve elements of both healthy and diseased plants. When fractured transversely, the sieve-tube walls usually had a distinctly lamellated appearance (Fig. 3). In both freeze-etched and embedded material, mycoplasmas were observed only in sieve elements of diseased plants and never in the healthy plants. The ultrastructural appearance of the mycoplasmas was similar to that of the aster yellows agent (Hirumi and Maramorosch, 1973). In thin sections the organisms showed great variability in shape and cytoplasmic density (Figs. 7-10). In those cells having a more electron-lucent cytoplasm, ribosomes, and DNA fibrils were easily seen. The mycoplasma plasmalemma averaged 9.5 nm in thickness (mean of 15 measurements).
V. rosea
PHLOEM
45
Under the conditions of specimen preparation used here, mycoplasmas showed a marked tendency to cross-fracture (Fig. 3). No details of the cytoplasmic matrix could be distinguished in cross-fractured organisms. The appearance of their cytoplasm was variable and depended on the quality of freezing. The fracture faces of the mycoplasma plasmalemmas were easily distinguished from those of sieve element plasmalemma invaginations (Fig. 2) and plastids (Fig. 5). The fracture faces of these structures had a much lower particle density than the mycoplasma cell membranes. However, mycoplasma membranes were not easily differentiated from the fracture faces of the outer mitochondrial membrane. In thin sections, mitochondria were always observed in a parietal position in both healthy and infected plants. For this reason, all organisms chosen for particle size and density determinations were situated in the central portion of the sieve tube lumen. Particle diameter ranged from 8.7 to 13.2 nm with a mean value of 10.8 nm. Particle densities varied from 682 to 1677 particles/~m 2. No consistent differences were noted in either particle size or density between protoplasmic and extracellular fracture faces of the mycoplasma plasmalemmas, and particles were usually evenly distributed on both fracture faces (Figs. 4, 5, 6). Within the cross-fractured mycoplasmas, intracellular vesicles were often observed (Figs. 4, 5, 11, 12). The fracture faces of these vesicle membranes had an unusual appearance in which irregularly shaped, smooth regions of the membrane 16 to 24 nm in diameter were demarcated by furrows. These vesicles were variable in shape and similar in size to intracellular vesicles observed in thin-sectioned mycoplasmas (Fig. 9). Occasionally, the intracellular vesicles appeared closely associated with the plasmalemma of the para-
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MYCOPLASMAS IN V. rosea PHLOEM site. A l t h o u g h vesicles of this t y p e probably arise as i n v a g i n a t i o n s of t h e p l a s m a l e m m a , serial sections indicated t h a t m o s t of the vesicles w e r e t r u l y i n t r a c e l l u l a r a n d not continuous w i t h the cell m e m b r a n e (Figs. 7-10). E x a m i n a t i o n of serial sections also showed t h a t m o s t m y c o p l a s m a s which c o n t a i n e d vesicles h a d one, or rarely, two per cell. The f r e q u e n c y of cells c o n t a i n i n g vesicles was e s t i m a t e d by e x a m i n i n g 10 sets of serial sections. E a c h series c o n t a i n e d 5 to 8 sections which were a p p r o x i m a t e l y 70-80 n m in thickness. Only those cells which were p r e s e n t in t h e central section of the series were scored for the presence or absence of vesicles. T h e entire series was t h e n e x a m i n e d a n d it was found t h a t 5 to 57% of the m y c o p l a s m a s c o n t a i n e d vesicles. Since m a n y of the large m y c o p l a s m a s which were p r e s e n t in the c e n t r a l section were not wholly w i t h i n t h e series, the percentages o b t a i n e d a r e c o n s e r v a t i v e estimates. T h e r e was a g e n e r a l correlation b e t w e e n i n c r e a s i n g m y c o p l a s m a population density w i t h i n a sieve e l e m e n t and the p e r c e n t a g e of m y c o p l a s m a s w i t h vesicles. In two p o p u l a t i o n s a s s u m e d to be senescent (characterized by swollen cells w i t h a v e r y electron-lucent p r o t o p l a s m a n d few nucleic acid strands) vesicles were found in 44 a n d 57% of t h e cells. Memb r a n e f r a c t u r e faces s i m i l a r to those of t h e i n t r a c e l l u l a r vesicleS, b u t not w i t h i n mycoplasmas, were often seen in sieve elem e n t s which w e r e densely p a c k e d w i t h senescent m y c o p l a s m a s (Figs. 11 a n d 12), I n t h i n sections, vesicle s h a p e was quite
47
v a r i a b l e r a n g i n g f r o m spherical forms to those which r e s e m b l e d two c l ~ e l y appressed m e m b r a n e s . Vesicles were not seen in small cells w i t h v e r y dense cytoplasm. M y c o p l a s m a s in freeze-etched m a t e r i a l were p r e d o m i n a n t l y circular or oval in s h a p e irrespective of w h e t h e r sieve elem e n t s were f r a c t u r e d l o n g i t u d i n a l l y or t r a n s v e r s e l y . The i r r e g u l a r a m o e b o i d s h a p e s of m y c o p l a s m a s , often observed in thin-sectioned m a t e r i a l , were not seen in freeze-etched m a t e r i a l . N a r r o w filamentous f o r m s were seen in freeze-etched m a terial only in the v i n c i n i t y of sieve p l a t e pores (Fig. 6). Such o r g a n i s m s could h a v e b e e n distorted d u r i n g p a s s a g e t h r o u g h the pores. Wider f i l a m e n t o u s f o r m s v~ere occasionally found in b o t h freeze-etched (Fig. 4) a n d sectioned m a t e r i a l (Fig. 13). F i l a m e n tous f o r m s were m o r e easily d e m o n s t r a t e d w h e n 0.5 t~m sections were e x a m i n e d . DISCUSSION In this study, particle d i a m e t e r s in the m y c o p l a s m a p l a s m a l e m m a s r a n g e d from 8.7 to 13.2 nm. Particles of a s i m i l a r size w e r e r e p o r t e d in the p l a s m a l e m m a of A c h o l e p l a s m a laidlawii (7.5 to 15 nm) (Tillack et al., 1970) a n d Mycoplasma meleagridis (6.6 to 14.4 nm) (Green a n d H a n son, 1973). The cell m e m b r a n e of Spirop l a s m a cirri, a p l a n t p a t h o g e n i c mollicute w i t h a helical morphology, contains particles a p p r o x i m a t e l y 10 n m in d i a m e t e r (Razin et al., 1973). Membrane systems generally have a
Fins. 1-6, 11, 12. Encircled arrow in freeze-etch micrographs indicates the direction of shadowing. FIG. 1. Transverse fracture of a sieve element (S) and companion cell (CC) from a healthy V. rosea plant. The lumen of the sieve element is free of organelles. A mitochondrion (mt) appears in a parietal position, pd, plasmodesma. Scale bar = 1 ~m. × 25 000. FIG. 2. Oblique-longitudinal fracture of a sieve element from a healthy plant. A plasmalemma invagination (pm) and endoplasmic" reticulum (er) can be seen adjacent to the cell wall (cw). Scale bar = 0.5 t~m. x 60 000. FIG. 3. Transverse fracture through a phloem bundle from a diseased plant. Sieve elements (S) contain plastids (p) and variable numbers of mycoplasmas, some of which are labeled (m). Scale bar = 1 t~m. × 15 000.
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M Y C O P L A S M A S IN V . r o s e a P H L O E M
greater particle density on the protoplasmic fracture face (Branton, 1969), and recent work indicates t h a t this is true for mycoplasmas (Green and Hanson, 1973; Razin et al., 1973; Tillack et al., 1970). This asymmetry could not be confirmed for the mycoplasmas in V. rosea, because of the variability in the range of particle densities on the pf and ef. A study of complementary fracture faces from the same organism could demonstrate unequivocally whether or not the membrane is asymmetric. A wide range of particle densities is not surprising in view of the general variability in the ultrastructural appearance of the mycoplasmas in V. rosea sieve elements. It seems likely t h a t all stages of the cell cycle are present in such heterogeneous populations and it has been suggested that particle density may vary with growth conditions and stages of the cell cycle (Branton, 1969). Amar et al: (1976) reported that Mycoplasma hominis membranes showed a marked increase in protein to lipid ratio as cultures of the organism aged. There has been some disagreement concerning the presence of membrane-bound vesicles (vacuoles) in mycoplasmas. Some authors feel that intracellular vesicles do not exist and that such observations are the result of misinterpretations of sections which have passed through invaginations in the mycoplasma plasmalemma (Black et al., 1972; Razin, 1969). Membranebound vesicles have been observed in several plant pathogenic mycoplasmas and recently Green and Hanson (1973) observed vesicles within an animal patho-
49
gen, Mycoplasma meleagridis. When M. meleagridis cultures were grown in the presence of potassium tellurite, the cell membranes were stained asymmetrically. Since both the plasmalemma and the vesicles were more heavily stained on the outer track of their unit membrane structure, the authors reasoned that the vesicles were intracellular and not plasmalemma invaginations. In the present work, serial sectioning has demonstrated that the vesicles in plant pathogenic mycoplasmas are intracellular as well. AIthough Green and Hanson used freezeetching in their study of M. meleagridis, their material fractured only through the plasmalemma and the structure of the vesicle membranes was not revealed. The significance of the unusual appearance of the vesicle fracture faces is unknown. Membranes described as having a "low profile bumpiness" have been observed in non-helical, bleb regions of S. citri (Cole et al., 1973). The criteria of Maniloff (1970) were used in identifying senescent mycoplasma populations. He studied the ultrastructure of A. laidlawii during culture development and found that death phase cultures contained rounded, swollen cells which had lost some of their intracytoplasmic material. He also noted some abnormal structures (intracytoplasmic membranes, dense membrane-bound inclusions, and empty extracellular membranous vesicles) which were probably the result of cellular degeneration. In the present work, the intracellular vesicles and the extracellular membranes
FIG. 4. M y c o p l a s m a s (m) in t h e l u m e n of a sieve e l e m e n t . Two of t h e o r g a n i s m s h a v e b e e n f r a c t u r e d to r e v e a l t h e e x t r a c e l l u l a r f r a c t u r e face (ef). T h e o t h e r o r g a n i s m is c r o s s - f r a c t u r e d a n d c o n t a i n s a vesicle (v). Scale b a r = 0.5 t~m. x 60 000. T h e i n s e t s h o w s t h e s a m e vesicle at × 126 000. FIG. 5. M y c o p l a s m a s (m) a n d a p l a s t i d (p) in a sieve e l e m e n t . P r o t o p l a s m i c f r a c t u r e face (pf) is s e e n in one o r g a n i s m , w h i l e t h e o t h e r is c r o s s - f r a c t u r e d r e v e a l i n g a vesicle (v). Scale b a r = 0.5 t~m. x 51 000. FIG. 6. L o n g i t u d i n a l f r a c t u r e t h r o u g h two sieve e l e m e n t s in t h e vicinity of a sieve plate. A sieve p l a t e pore (po) is in t h e p l a n e of f r a c t u r e . M y c o p l a s m a s (m) a r e p r e s e n t in b o t h sieve e l e m e n t s . A r r o w s i n d i c a t e e l o n g a t e d m e m b r a n e s w h i c h m a y be portions of m y c o p l a s m a s w h i c h were d i s t o r t e d d u r i n g p a s s a g e t h r o u g h t h e pore. cw, cell wall. Scale b a r = 0.5 ~ m . x 50 000.
|
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MYCOPLASMAS IN V. rosea PHLOEM
51
with the same unusual membrane structure were most frequently associated with senescent pathogen populations. It seems likely that they may be products of cellular degeneration of the mycoplasmas. There is little doubt that mycoplasma morphology varies with both environmental conditions and the growth cycle of the organisms (Boatman, 1973; Maniloff, 1970). It is also well established that the osmolality of fixatives used in electron microscopy can alter the morphology and ultrastructure of mycoplasmas (Lemcke, 1972). However, the mycoplasmas in thin sections of V . r o s e a plants often showed constrictions and amoeboid shapes that were not apparent in freeze-etch preparations and it seems possible that some of the steps following glutaraldehyde fixation were responsible for such forms. Fineran (1970) has shown that plant vacuoles were predominantly spherical in freeze-etched tissue even if it was prefixed with glutaraldehyde, while sectioned vacuoles were more variable in shape. A similar situation could be occurring with mycoplasmas in plant tissues that are fixed and embedded for electron microscopy.
for use of their freeze-etching facilities, and Drs. J. R. Aist and M. V. P a r t h a s a r a t h y , and R. J. Howard for their helpful discussions during the course of this work. Portions of this work were made possible by CSRS g r a n t No. 316-15-53 awarded to J. R. Aist and H. W. Israel.
I would like to thank the section of Genetics, Development, and Physiology, Cornell University,
TILLACK, T. W., CARTER, R., AND RAZIN, S., BiG-
REFERENCES AMAR, A., ROTTEM,S., KAHANE,I., AND RAZIN, S., Biochem. Biophys. Acta 426, 258 (1976). BLACK, F. T., BIRCH-ANDERSON, A., AND FREUNDT, E. A., J. Bacteriol. 111, 254 (1972). BOATMAN, E., Ann. N . Y . Acad. Sci. 225, 172 (1973). BRANTON, D., A n n u . Rev. Plant Physiol. 20, 209 (1969). BRANTON, D., BULLIVANT,S., GILULA,N. B., KAR-
NOVSKY, M. J., MOOR, H., MULETHALER, K., NORTHCOTE,D. H., PACKER,L., SATIR, B., SATIR, P., SPETH, V., STAEHLIN, L. A., STEERE, R. L., AND WEINSTEIN,R. S., Science 190, 54 (1975). COLE, R. M., TULLY, J. G., POPKIN, T. J., AND BOVE, J. M., Ann. N . Y . Acad. Sci. 225, 471 (1973). FINERAN, B. A., Protoplasma 70, 457 (1970). GREEN, F., AND HANSON, R. P., J. Bacteriol. 116, 1011 (1973). HIRUMI, H., AND MARAMOROSCH, K., Ann. N. Y. Acad. Sci. 225, 201 (1973). LEMCKE, a. M., J. Bacteriol. 110, 1154 (1972). MANILOFF, J., J. Bacteriol. 102, 561 (1970). MOOR,H., ANDMULETHALER,K . , J . CellBiol. 17, 609 (1963). RAZIN, S., Annu. Rev. Microbiol. 23, 317 (1969).
RAZiN, S., HASIN,M., NE'EMAN,Z., ANDROTTEM,S., J. Bacteriol. 116, 1421 (1973). chem. Biophys. Acta 219, 123 (1970).
FIGS. 7-10. Adjacent serial sections through mycoplasmas illustrating the intracellular n a t u r e of the vesicle (arrow in Fig. 9). There is no continuity between the vesicle m e m b r a n e and the plasmalemma of the mycoplasma. Scale bar = 0.5 tLm. × 40 000. FIG. 11. Transverse fracture through a sieve element which is densely packed with mycoplasmas. The arrow indicates a n extracellular m e m b r a n e w h i c h ' h a s the same unusual structure as the intracellular mycoplasma vesicle (v). Scale b a r = 0.5 t~m. × 44 000. FIG. 12. Extracellular m e m b r a n e with unusual m e m b r a n e structure (arrow) and vesicle (v) within the l u m e n of a sieve element which is densely packed with mycoplasmas. Scale b a r = 0.25 t~m. × 74 000. FIG. 13. Thick (0.5 t~m) section showing several filamentous mycoplasmas, cw, cell wall. Scale bar = 0.5 t~m. × 34 000.
(1977)
A Freeze-Etch and Thin-Section Study of Mycoplasmas in Vinca rosea Phloem E. J. BRAUN Department of Plant Pathology, Cornell University, Ithaca, New York 14853 ~Received November 12, 1976, and in revised form, March 7, 1977 Mycoplasmas in the phloem of V. rosea were examined in thin sections and freeze-etchings. The plasmalemma of the parasite possessed evenly distributed particles on both fracture faces; particie diameters ranged from 8.7 to 13.2 nm. Vesicles in some mycoplasmas were found, in serial sections, to be wholly intracellular. Vesicles occurred most frequently in mycoplasmas thought to be senescent (characterized by swollen cells with very electron-lucent cytoplasm and a few nucleic acid strands). The fracture faces of the vesicular membranes lacked particles, and irregularly shaped regions 16 to 24 nm in diameter were demarcated by furrows. The functional significance of this unusual membrane structure was not determined.
Plant pathogenic mycoplasmas are transmitted by phloem-feeding insects and are confined to the phloem tissue in infected plants. Intracellular vesicles have been seen in many plant pathogenic mycoplasmas and they have been described in some detail by Hirumi and Maramorosch (1973). These authors, who worked with the aster yellows agent, referred to such structures as vacuoles. The present study was undertaken to gain more information regarding the structure of plant pathogenic mycoplasmas, particularly that of their intracellular vesicles.
ferred to a refrigerator (4°C) for an additional 5 hr. After washing in buffer, the material was postfixed in 1% OsO~ in the same buffer for 12-18 hr at 4°C. Tissue was dehydrated in an acetone series and embedded in Epon-Araldite. Thin and thick (0.5 #m) sections were made with glass knives on a LKB-Huxley ultramicrotome. Serial thin sections were picked up on 100 mesh Formvar-coated grids. Freeze-etching. Samples comparable to those used for thin sectioning were excised in a similar manner and fixed in 3% glutaraldehyde in 0.08 M phosphate buffer (pH 7.0) at room temperature for 1 hr. They were then washed in buffer, infiltrated with 20 or 30% glycerol, and frozen in liquid Freon-22 cooled in liquid nitrogen. The specimens were fractured at -101°C in a Balzers freeze-etching machine (BA 360M) and shadowed with platinum and carbon according to the method of Moor and Mulethaler (1963). Replicas were cleaned with 70% H2SO4 (18 hr), 40% chromic acid (18 hr), and 70% H~SO4 (6 hr). Before and after the final H2SO4 treatment, replicas were transferred to bleach for 1-2 hr. The replicas were picked up on Formvar-coated grids. The terminology of Branton et al. (1975) has been used in identifying fracture faces. The fracture face of the membrane which is closest to the cytoplasm is termed the protoplasmic fracture face (pf) and that closest to the extracellular or endoplasmic space is termed the extracellular or endoplasmic fracture face (ef). Particle size and density on membrane fracture faces of the mycoplasmas were determined by taking data from 10 protoplasmic and 10 extracellular fracture faces. Maximum shadow width of 12 randomly selected particles were measured on each fracture face. Density was determined by counting particles within three randomly selected 1 cm ~ areas on each
MATERIALS AND METHODS Mycoplasmas were transmitted via dodder (Cuscuta epithymum Murr.) from phloem necrosis-diseased American elm (Ulmus americana L.) seedlings to periwinkle plants (Vinca rosea L.). Samples (petioles and axillary shoots) were taken from V. rosea plants showing advanced stages of symptom development characterized by extensive proliferation of axillary shoots, stunted, chlorotic leaves, and cessation of flowering. Samples of healthy tissue (petioles and midribs) were obtained from V. rosea plants grown from seed. Both diseased and healthy plants were grown under greenhouse conditions. Thin sectioning. Samples were excised in a petri dish containing 3% glutaraldehyde in 0.08 M phosphate buffer (pH 7.0). The central portion of each sample was transferred to fresh fixative and evacuated in a desiccator for 5-10 min. After 1 hr at room temperature the material in fixative was trans44 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
MYCOPLASMAS IN fracture face. All measurements were made on micrographs at a magnificationof x 90 000. Replicas and thin sections were observed and photographed with a Philips EM 201 at 80 kV; 0.5 t~m sections were viewed at 100 kV. RESULTS In V . r o s e a the phloem occurs in bundles or strands consisting of sieve-tube elements, companion cells, and phloem parenchyma cells. Phloem develops both externally and internally with respect to the xylem. Structurally, the bundles of external and internal phloem are very similar and no attempt was made to differentiate between them in this study. Phloem was recongized in freeze-etch preparations by its spatial relationship to the xylem and because its cells are narrower t h a n those of the surrounding vascular parenchyma and cortex. Sieve-tube elements can be distinguished from the surrounding cells by several criteria. In healthy plants the lumen of the sieve element Was always free oforganelles (Figs. 1 and 2). Mitochondria, plastids, and endoplasmic reticulum were confined to the parietal region of the sieve element protoplast. Invaginations of the plasmalemma (Fig. 2) were common in sieve elements of both healthy and diseased plants. When fractured transversely, the sieve-tube walls usually had a distinctly lamellated appearance (Fig. 3). In both freeze-etched and embedded material, mycoplasmas were observed only in sieve elements of diseased plants and never in the healthy plants. The ultrastructural appearance of the mycoplasmas was similar to that of the aster yellows agent (Hirumi and Maramorosch, 1973). In thin sections the organisms showed great variability in shape and cytoplasmic density (Figs. 7-10). In those cells having a more electron-lucent cytoplasm, ribosomes, and DNA fibrils were easily seen. The mycoplasma plasmalemma averaged 9.5 nm in thickness (mean of 15 measurements).
V. rosea
PHLOEM
45
Under the conditions of specimen preparation used here, mycoplasmas showed a marked tendency to cross-fracture (Fig. 3). No details of the cytoplasmic matrix could be distinguished in cross-fractured organisms. The appearance of their cytoplasm was variable and depended on the quality of freezing. The fracture faces of the mycoplasma plasmalemmas were easily distinguished from those of sieve element plasmalemma invaginations (Fig. 2) and plastids (Fig. 5). The fracture faces of these structures had a much lower particle density than the mycoplasma cell membranes. However, mycoplasma membranes were not easily differentiated from the fracture faces of the outer mitochondrial membrane. In thin sections, mitochondria were always observed in a parietal position in both healthy and infected plants. For this reason, all organisms chosen for particle size and density determinations were situated in the central portion of the sieve tube lumen. Particle diameter ranged from 8.7 to 13.2 nm with a mean value of 10.8 nm. Particle densities varied from 682 to 1677 particles/~m 2. No consistent differences were noted in either particle size or density between protoplasmic and extracellular fracture faces of the mycoplasma plasmalemmas, and particles were usually evenly distributed on both fracture faces (Figs. 4, 5, 6). Within the cross-fractured mycoplasmas, intracellular vesicles were often observed (Figs. 4, 5, 11, 12). The fracture faces of these vesicle membranes had an unusual appearance in which irregularly shaped, smooth regions of the membrane 16 to 24 nm in diameter were demarcated by furrows. These vesicles were variable in shape and similar in size to intracellular vesicles observed in thin-sectioned mycoplasmas (Fig. 9). Occasionally, the intracellular vesicles appeared closely associated with the plasmalemma of the para-
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MYCOPLASMAS IN V. rosea PHLOEM site. A l t h o u g h vesicles of this t y p e probably arise as i n v a g i n a t i o n s of t h e p l a s m a l e m m a , serial sections indicated t h a t m o s t of the vesicles w e r e t r u l y i n t r a c e l l u l a r a n d not continuous w i t h the cell m e m b r a n e (Figs. 7-10). E x a m i n a t i o n of serial sections also showed t h a t m o s t m y c o p l a s m a s which c o n t a i n e d vesicles h a d one, or rarely, two per cell. The f r e q u e n c y of cells c o n t a i n i n g vesicles was e s t i m a t e d by e x a m i n i n g 10 sets of serial sections. E a c h series c o n t a i n e d 5 to 8 sections which were a p p r o x i m a t e l y 70-80 n m in thickness. Only those cells which were p r e s e n t in t h e central section of the series were scored for the presence or absence of vesicles. T h e entire series was t h e n e x a m i n e d a n d it was found t h a t 5 to 57% of the m y c o p l a s m a s c o n t a i n e d vesicles. Since m a n y of the large m y c o p l a s m a s which were p r e s e n t in the c e n t r a l section were not wholly w i t h i n t h e series, the percentages o b t a i n e d a r e c o n s e r v a t i v e estimates. T h e r e was a g e n e r a l correlation b e t w e e n i n c r e a s i n g m y c o p l a s m a population density w i t h i n a sieve e l e m e n t and the p e r c e n t a g e of m y c o p l a s m a s w i t h vesicles. In two p o p u l a t i o n s a s s u m e d to be senescent (characterized by swollen cells w i t h a v e r y electron-lucent p r o t o p l a s m a n d few nucleic acid strands) vesicles were found in 44 a n d 57% of t h e cells. Memb r a n e f r a c t u r e faces s i m i l a r to those of t h e i n t r a c e l l u l a r vesicleS, b u t not w i t h i n mycoplasmas, were often seen in sieve elem e n t s which w e r e densely p a c k e d w i t h senescent m y c o p l a s m a s (Figs. 11 a n d 12), I n t h i n sections, vesicle s h a p e was quite
47
v a r i a b l e r a n g i n g f r o m spherical forms to those which r e s e m b l e d two c l ~ e l y appressed m e m b r a n e s . Vesicles were not seen in small cells w i t h v e r y dense cytoplasm. M y c o p l a s m a s in freeze-etched m a t e r i a l were p r e d o m i n a n t l y circular or oval in s h a p e irrespective of w h e t h e r sieve elem e n t s were f r a c t u r e d l o n g i t u d i n a l l y or t r a n s v e r s e l y . The i r r e g u l a r a m o e b o i d s h a p e s of m y c o p l a s m a s , often observed in thin-sectioned m a t e r i a l , were not seen in freeze-etched m a t e r i a l . N a r r o w filamentous f o r m s were seen in freeze-etched m a terial only in the v i n c i n i t y of sieve p l a t e pores (Fig. 6). Such o r g a n i s m s could h a v e b e e n distorted d u r i n g p a s s a g e t h r o u g h the pores. Wider f i l a m e n t o u s f o r m s v~ere occasionally found in b o t h freeze-etched (Fig. 4) a n d sectioned m a t e r i a l (Fig. 13). F i l a m e n tous f o r m s were m o r e easily d e m o n s t r a t e d w h e n 0.5 t~m sections were e x a m i n e d . DISCUSSION In this study, particle d i a m e t e r s in the m y c o p l a s m a p l a s m a l e m m a s r a n g e d from 8.7 to 13.2 nm. Particles of a s i m i l a r size w e r e r e p o r t e d in the p l a s m a l e m m a of A c h o l e p l a s m a laidlawii (7.5 to 15 nm) (Tillack et al., 1970) a n d Mycoplasma meleagridis (6.6 to 14.4 nm) (Green a n d H a n son, 1973). The cell m e m b r a n e of Spirop l a s m a cirri, a p l a n t p a t h o g e n i c mollicute w i t h a helical morphology, contains particles a p p r o x i m a t e l y 10 n m in d i a m e t e r (Razin et al., 1973). Membrane systems generally have a
Fins. 1-6, 11, 12. Encircled arrow in freeze-etch micrographs indicates the direction of shadowing. FIG. 1. Transverse fracture of a sieve element (S) and companion cell (CC) from a healthy V. rosea plant. The lumen of the sieve element is free of organelles. A mitochondrion (mt) appears in a parietal position, pd, plasmodesma. Scale bar = 1 ~m. × 25 000. FIG. 2. Oblique-longitudinal fracture of a sieve element from a healthy plant. A plasmalemma invagination (pm) and endoplasmic" reticulum (er) can be seen adjacent to the cell wall (cw). Scale bar = 0.5 t~m. x 60 000. FIG. 3. Transverse fracture through a phloem bundle from a diseased plant. Sieve elements (S) contain plastids (p) and variable numbers of mycoplasmas, some of which are labeled (m). Scale bar = 1 t~m. × 15 000.
:Z
Qo
M Y C O P L A S M A S IN V . r o s e a P H L O E M
greater particle density on the protoplasmic fracture face (Branton, 1969), and recent work indicates t h a t this is true for mycoplasmas (Green and Hanson, 1973; Razin et al., 1973; Tillack et al., 1970). This asymmetry could not be confirmed for the mycoplasmas in V. rosea, because of the variability in the range of particle densities on the pf and ef. A study of complementary fracture faces from the same organism could demonstrate unequivocally whether or not the membrane is asymmetric. A wide range of particle densities is not surprising in view of the general variability in the ultrastructural appearance of the mycoplasmas in V. rosea sieve elements. It seems likely t h a t all stages of the cell cycle are present in such heterogeneous populations and it has been suggested that particle density may vary with growth conditions and stages of the cell cycle (Branton, 1969). Amar et al: (1976) reported that Mycoplasma hominis membranes showed a marked increase in protein to lipid ratio as cultures of the organism aged. There has been some disagreement concerning the presence of membrane-bound vesicles (vacuoles) in mycoplasmas. Some authors feel that intracellular vesicles do not exist and that such observations are the result of misinterpretations of sections which have passed through invaginations in the mycoplasma plasmalemma (Black et al., 1972; Razin, 1969). Membranebound vesicles have been observed in several plant pathogenic mycoplasmas and recently Green and Hanson (1973) observed vesicles within an animal patho-
49
gen, Mycoplasma meleagridis. When M. meleagridis cultures were grown in the presence of potassium tellurite, the cell membranes were stained asymmetrically. Since both the plasmalemma and the vesicles were more heavily stained on the outer track of their unit membrane structure, the authors reasoned that the vesicles were intracellular and not plasmalemma invaginations. In the present work, serial sectioning has demonstrated that the vesicles in plant pathogenic mycoplasmas are intracellular as well. AIthough Green and Hanson used freezeetching in their study of M. meleagridis, their material fractured only through the plasmalemma and the structure of the vesicle membranes was not revealed. The significance of the unusual appearance of the vesicle fracture faces is unknown. Membranes described as having a "low profile bumpiness" have been observed in non-helical, bleb regions of S. citri (Cole et al., 1973). The criteria of Maniloff (1970) were used in identifying senescent mycoplasma populations. He studied the ultrastructure of A. laidlawii during culture development and found that death phase cultures contained rounded, swollen cells which had lost some of their intracytoplasmic material. He also noted some abnormal structures (intracytoplasmic membranes, dense membrane-bound inclusions, and empty extracellular membranous vesicles) which were probably the result of cellular degeneration. In the present work, the intracellular vesicles and the extracellular membranes
FIG. 4. M y c o p l a s m a s (m) in t h e l u m e n of a sieve e l e m e n t . Two of t h e o r g a n i s m s h a v e b e e n f r a c t u r e d to r e v e a l t h e e x t r a c e l l u l a r f r a c t u r e face (ef). T h e o t h e r o r g a n i s m is c r o s s - f r a c t u r e d a n d c o n t a i n s a vesicle (v). Scale b a r = 0.5 t~m. x 60 000. T h e i n s e t s h o w s t h e s a m e vesicle at × 126 000. FIG. 5. M y c o p l a s m a s (m) a n d a p l a s t i d (p) in a sieve e l e m e n t . P r o t o p l a s m i c f r a c t u r e face (pf) is s e e n in one o r g a n i s m , w h i l e t h e o t h e r is c r o s s - f r a c t u r e d r e v e a l i n g a vesicle (v). Scale b a r = 0.5 t~m. x 51 000. FIG. 6. L o n g i t u d i n a l f r a c t u r e t h r o u g h two sieve e l e m e n t s in t h e vicinity of a sieve plate. A sieve p l a t e pore (po) is in t h e p l a n e of f r a c t u r e . M y c o p l a s m a s (m) a r e p r e s e n t in b o t h sieve e l e m e n t s . A r r o w s i n d i c a t e e l o n g a t e d m e m b r a n e s w h i c h m a y be portions of m y c o p l a s m a s w h i c h were d i s t o r t e d d u r i n g p a s s a g e t h r o u g h t h e pore. cw, cell wall. Scale b a r = 0.5 ~ m . x 50 000.
|
F
MYCOPLASMAS IN V. rosea PHLOEM
51
with the same unusual membrane structure were most frequently associated with senescent pathogen populations. It seems likely that they may be products of cellular degeneration of the mycoplasmas. There is little doubt that mycoplasma morphology varies with both environmental conditions and the growth cycle of the organisms (Boatman, 1973; Maniloff, 1970). It is also well established that the osmolality of fixatives used in electron microscopy can alter the morphology and ultrastructure of mycoplasmas (Lemcke, 1972). However, the mycoplasmas in thin sections of V . r o s e a plants often showed constrictions and amoeboid shapes that were not apparent in freeze-etch preparations and it seems possible that some of the steps following glutaraldehyde fixation were responsible for such forms. Fineran (1970) has shown that plant vacuoles were predominantly spherical in freeze-etched tissue even if it was prefixed with glutaraldehyde, while sectioned vacuoles were more variable in shape. A similar situation could be occurring with mycoplasmas in plant tissues that are fixed and embedded for electron microscopy.
for use of their freeze-etching facilities, and Drs. J. R. Aist and M. V. P a r t h a s a r a t h y , and R. J. Howard for their helpful discussions during the course of this work. Portions of this work were made possible by CSRS g r a n t No. 316-15-53 awarded to J. R. Aist and H. W. Israel.
I would like to thank the section of Genetics, Development, and Physiology, Cornell University,
TILLACK, T. W., CARTER, R., AND RAZIN, S., BiG-
REFERENCES AMAR, A., ROTTEM,S., KAHANE,I., AND RAZIN, S., Biochem. Biophys. Acta 426, 258 (1976). BLACK, F. T., BIRCH-ANDERSON, A., AND FREUNDT, E. A., J. Bacteriol. 111, 254 (1972). BOATMAN, E., Ann. N . Y . Acad. Sci. 225, 172 (1973). BRANTON, D., A n n u . Rev. Plant Physiol. 20, 209 (1969). BRANTON, D., BULLIVANT,S., GILULA,N. B., KAR-
NOVSKY, M. J., MOOR, H., MULETHALER, K., NORTHCOTE,D. H., PACKER,L., SATIR, B., SATIR, P., SPETH, V., STAEHLIN, L. A., STEERE, R. L., AND WEINSTEIN,R. S., Science 190, 54 (1975). COLE, R. M., TULLY, J. G., POPKIN, T. J., AND BOVE, J. M., Ann. N . Y . Acad. Sci. 225, 471 (1973). FINERAN, B. A., Protoplasma 70, 457 (1970). GREEN, F., AND HANSON, R. P., J. Bacteriol. 116, 1011 (1973). HIRUMI, H., AND MARAMOROSCH, K., Ann. N. Y. Acad. Sci. 225, 201 (1973). LEMCKE, a. M., J. Bacteriol. 110, 1154 (1972). MANILOFF, J., J. Bacteriol. 102, 561 (1970). MOOR,H., ANDMULETHALER,K . , J . CellBiol. 17, 609 (1963). RAZIN, S., Annu. Rev. Microbiol. 23, 317 (1969).
RAZiN, S., HASIN,M., NE'EMAN,Z., ANDROTTEM,S., J. Bacteriol. 116, 1421 (1973). chem. Biophys. Acta 219, 123 (1970).
FIGS. 7-10. Adjacent serial sections through mycoplasmas illustrating the intracellular n a t u r e of the vesicle (arrow in Fig. 9). There is no continuity between the vesicle m e m b r a n e and the plasmalemma of the mycoplasma. Scale bar = 0.5 tLm. × 40 000. FIG. 11. Transverse fracture through a sieve element which is densely packed with mycoplasmas. The arrow indicates a n extracellular m e m b r a n e w h i c h ' h a s the same unusual structure as the intracellular mycoplasma vesicle (v). Scale b a r = 0.5 t~m. × 44 000. FIG. 12. Extracellular m e m b r a n e with unusual m e m b r a n e structure (arrow) and vesicle (v) within the l u m e n of a sieve element which is densely packed with mycoplasmas. Scale b a r = 0.25 t~m. × 74 000. FIG. 13. Thick (0.5 t~m) section showing several filamentous mycoplasmas, cw, cell wall. Scale bar = 0.5 t~m. × 34 000.
